Summary The long term goal of this research project is to understand the molecular regulatory mechanism of TGF-? signaling during palatogenesis. Cleft palate represents one of the major groups of congenital birth defects in the human population. Importantly, we and others have shown that mutations in TGF-? signaling can cause cleft palate in both mice and humans. Specifically, TGF-? is required for inducing apoptosis in the medial edge epithelium (MEE) and cell proliferation in the cranial neural crest (CNC)-derived palatal mesenchyme, both of which are crucial for normal palatal fusion. In this competing renewal application, we will carry out experiments to test the hypotheses that TGF-? signaling mediator Smad4, its downstream target Msx1 and the interaction between Smad4 and Msx1 are crucial for the cell fate determination of CNC-derived palatal mesenchyme. Furthermore, TGF-? regulates the expression of downstream target genes, such as Irf6 and Ctgf, to control the fate of MEE and CNC cells during palatogenesis. We have proposed three specific aims. In Specific Aim 1, we will investigate the functional significance of Smad4 in regulating the fate of CNC- derived palatal mesenchyme during palatogenesis. Furthermore, we will test the hypotheses that there is a CNC cell-autonomous requirement for Msx1 signaling during palatogenesis and Smad4/Msx1 interaction is crucial for regulating the fate of CNC-derived palatal mesenchyme. In Specific Aim 2, we will investigate the functional significance of TGF-? mediated Irf6 signaling in controlling the fate of MEE cells during palatal fusion. We will test the hypotheses that TGF-? is responsible for inducing Irf6 expression in MEE cells, Irf6 is required for MEE cells to undergo apoptosis, and TGF-?-mediated Irf6 expression is critical for apoptosis in MEE cells and for normal palatal fusion. In Specific Aim 3, we will explore the biological significance of TGF-? signaling mediated Ctgf expression in regulating CNC cell proliferation during palatogenesis. We will test the hypotheses that TGF-?-induced Ctgf expression is required for CNC cell proliferation in the developing palatal shelf and TGF-?-mediated CTGF signaling is critical for palatogenesis. Ultimately, this study will provide a better understanding of how the TGF-? signaling cascade regulates palatogenesis and will lead to the development of methods for better diagnosis, treatment and prevention of cleft palate.